Membrane and proteome allocation constraints in Escherichia coli models during overflow metabolism

The allocation of finite cellular resources is a fundamental principle that dictates microbial metabolic strategies and gives rise to complex phenomena, such as overflow metabolism, characterized by the production of respiro-fermentative by-products, including acetate, during rapid growth. Although proteome-constrained models have successfully predicted overflow metabolism in Escherichia coli, they often overlook the distinct biophysical and energetic costs associated with protein localization. The cellular membrane, in particular, represents a critical and constrained compartment where competition for space and synthesis machinery can create significant metabolic bottlenecks. To investigate this, we developed the membrane-associated constrained flux balance analysis (MAFBA), a scalable, genome-scale metabolic model that introduces a tunable constraint on the total protein mass allocated to the cellular membrane. Our model demonstrates that the overall and membrane-associated proteome allocation constraints interact to improve the accuracy of predicting the onset of overflow metabolism. It mechanistically reveals that at high growth rates, competition for limited membrane allocation forces a trade-off between growth-essential functions and respiratory capacity, leading to acetate production. Furthermore, MAFBA quantitatively explains the widely observed experimental phenomenon that expressing heterologous membrane proteins imposes a significantly higher metabolic burden than expressing cytosolic proteins. This study establishes membrane resource allocation as a key constraint governing bacterial physiology, acting in concert with overall proteome limitations. The resulting MAFBA framework provides a powerful and accessible tool for synthetic biology and metabolic engineering, enabling the prediction of metabolic costs associated with expressing membrane-bound proteins and guiding strain design strategies, holding promise for applications in bioproduction and metabolic engineering.